Classically, vaccines against enveloped viruses either contain killed or live attenuated viruses, or contain their membrane proteins (e.g., split virus preparation). After injection, the virus particles or the proteins are taken up by cells of the immune system (for instance, dendritic cells or macrophages), followed by a presentation of their antigenic parts to effector cells of the immune system. Most vaccines have to be injected to elicit a sufficiently strong immune response because antigen-presenting phagocytes are most abundant just under the skin. However, it has now become clear that such cells are also present in the mucosa that, for instance, lines the nose (Ogra et al. 2001). The phagocytes of the mucosa require much stronger stimulation than those present under the skin (Janeway et al. 2001).
While the injection of some viruses or proteins, for example, influenza virus, elicits an immune response that is sufficiently strong to protect against a later infection by the same virus, this is not the case for many others, for example, respiratory syncytial virus. Numerous attempts to reinforce the immune response by physical or chemical means (by compounds called adjuvants) have been undertaken. The most important principles that emerge from these experiments are as follows. For physical stimulation, it has been found that particles containing multiple copies of viral subunits, such as whole viruses, virosomes, and proteins on microparticle carriers stimulate the immune system better than individual subunits (Ogra et al. 2001; Janeway et al. 2001), whereas chemical stimulation requires that the phagocytes or the effector cells of the immune system receive certain signals through receptors present on the cell surface, for instance, through the use of an adjuvant. With sufficient additional physicochemical stimulation, viral proteins can elicit strong immune responses even if applied to mucous membranes, for example, upon intranasal application (Ogra et al. 2001). Most of the current methods and compositions for stimulating an immune response by such means, whether by chemical or physical means or combinations of the two principles, have significant disadvantages that will be outlined below.
A particular kind of vaccine composition that was developed in the art is known as “virosomes,” which are lipid bilayers containing viral glycoproteins (FIG. 1). Virosomes are generally produced by extraction of membrane proteins from viruses with detergents, followed by removal of the detergent in the presence of lipids such that characteristic lipid bilayers are formed with the proteins protruding from them (Stegmann et al. 1987). For some viral antigens, such virosomes elicit protective immune responses that are strong even when the vaccine is delivered through intranasal application (as is exemplified in PCT International Patent Application Publications WO 88/08718 and WO 92/19267). However, 30 to 85% of the viral proteins are lost during the process of virosome formation (WO 88/08718; Stegmann et al. 1987). Moreover, as the insertion of viral protein on either side of the membrane during reconstitution occurs with approximately equal probability, a large part of the protein (one third in the case of the influenza hemagglutinin, Stegmann et al. 1987) present in virosomes is present on the inside of the particles, and thus invisible to the immune system. Also, it is well known in the art that such artificial lipid bilayers render the preparation fragile, causing storage, handling and transport problems. Moreover, optimal formulation often requires complex mixtures of lipids, whose ratio has to be strictly controlled during production. This poses regulatory problems.
The immunogenicity of many viral antigens, for example, influenza hemagglutinin, is only slightly improved with respect to killed viruses when the antigen is presented from virosomes (Gluck et al. 1994). Therefore, to enhance the immune response allowing intranasal application of this vaccine, an adjuvant protein from Escherichia coli (heat-labile toxin) was mixed with the virosome influenza vaccine (EP 0538437). Clinical trials indicated that addition of the toxin was necessary to induce serum antibody titers equivalent to injected vaccine (Gluck et al. 1994). Although addition of the toxin did thus enhance the immunogenicity, it also induced a serious side effect known as Bell's Palsy, a temporary paralysis of facial muscles. In this case, the toxin did not form part of the virosomes but was present in the surrounding solution. As the adjuvanting effect of the toxin is due to recognition by an antigen-presenting cell, enhancing its reaction to a viral protein that the cell might take up, and since there is no certainty that the toxin and the viral protein will contact the same cell, a relatively high concentration of the toxin needs to be used in order to ensure activation of every cell. Therefore, clearly, virosomes have promising features, such as their particulate nature, but a fair number of disadvantages.
Alternatively, researchers in the art have also generated antigen complexes different from virosomes, such as “Immunostimulatory Complexes” (ISCOMs, Morein et al. 1984), containing viral proteins complexed with compounds such as Quil A® and saponins (EP 0231039B1; EP 0109942A1; EP 0180564A1), predominantly isolated from the bark of Quillaia sopanaria Molina. Mixed with antigen and lipids such as cholesterol, these compounds form cage-like structures of between 30 to 40 nm, rendering the antigen particulate, while acting at the same time as an adjuvant. Although ISCOMs have been used in a number of veterinary vaccines and enhance the immunogenicity of the viral membrane proteins, the development of such vaccines for humans has been inhibited by concerns about their toxicity and the complexity of the mixture (Cox et al. 1998).
More recently, proteosome influenza vaccines were developed (U.S. Pat. No 6,743,900), consisting of non-covalent complexes of the purified outer membrane proteins of bacteria such as meningococci, mixed with antigenic proteins such as the influenza hemagglutinin or the human immunodeficiency envelope glycoprotein. While the presence of these multiple bacterial proteins may act as an adjuvant, the complex nature of such mixtures consisting of multiple proteins will present a regulatory issue.
Another particulate formulation developed by Biovector Therapeutics consists of an inner core of carbohydrate surrounded by a lipid envelope containing antigens. With influenza hemagglutinin as the antigen, some enhancement of the immune response was noted, but not significant enough to warrant further development.
Live attenuated versions of respiratory viruses, such as a cold-adapted strain of influenza virus with minimal replication in the respiratory tract have been developed as intranasal vaccines. These vaccines have the advantage of inducing immune responses that are close to the natural immunity induced by an infection with wild-type virus. For influenza, such vaccines have been known for 20 years and now appear close to commercialization. The delay has been caused by concerns about the ability of many viruses to mutate rapidly, causing the properties of attenuated viruses to revert partially or wholly to wild-type virus and, in fact, causing the disease they were meant to prevent.
For the above reasons, it is well recognized in the art that a need still exists for new vaccine compositions that induce a strong immune response, that do not have the disadvantages of live virus vaccines, that are easily applicable and that have low toxicity. It is, therefore, recognized in the art that there is a need for sub-unit vaccines for intranasal delivery.